key: cord-0758233-y1hjgmmv
authors: Zhang, Chunyun; Chen, Qixin; Liu, Fuguo; Liu, Yin; Wang, Yuanyuan; Chen, Guofu
title: Rapid detection of Chattonella marina by PCR combined with dot lateral flow strip
date: 2022-01-21
journal: J Appl Phycol
DOI: 10.1007/s10811-021-02667-x
sha: cf91c841f2af62e58de4d6a27c1c0628e665bbe6
doc_id: 758233
cord_uid: y1hjgmmv

In this study a novel technique referred to as PCR combined with dot lateral flow strip (PCDS) is proposed and its application to the detection of harmful microalgae was explored. For this purpose, using Chattonella marina as a test algal species, PCR targeting the D1–D2 region of large subunit ribosomal gene of this alga was performed with the tagged specific primers. The amplicons were then analyzed with the manually prepared dot lateral flow strip, and the strip could produce a test dot and a control dot that are naked-eye detectable, indicating the successful establishment of PCDS. The established PCDS assay does not require expensive instruments for the detection, and the results can be observed visually after adding 7.5 μL of PCR amplicons in combination with 92.5 μL of chromatography buffer to the sample pad of the strip for about 10 min. The PCR conditions were optimized to enhance the effectiveness of detection. The cross-reactivity test with 23 microalgae species, including Chattonella marina, showed good specificity of the PCDS. The detection limit of PCDS was 1.25 × 10(−2) ng µL(−1) for genomic DNA and 10(1) cells mL(−1) for crude cell extracts, which can meet the detection needs. In summary, the PCDS proposed in this study has low cost, clear, and intuitive detection results and good specificity and sensitivity, providing a novel detection method for C. marina. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10811-021-02667-x.

Harmful algal blooms (HABs) refer to natural phenomena of mass propagation of algal cells in a short period due to the eutrophication of seawater, which usually can cause massive mortality of aquatic animals (Foss et al. 2019) . In recent years HABs have become an increasingly serious environmental problem and pose a threat to human health (Huang et al. 2018 ) and sustainable development of the marine economy (Wang et al. 2017a) . Therefore, it is essential to establish effective and convenient techniques for the early detection of harmful algae to take timely and effective measures to reduce the potential economic damage caused by HABs.

Chattonella marina, as a member of Raphidophyceae, is a highly adaptable marine harmful microalga. This algal species can live in a wide range of temperatures (15-30 °C) and salinities (10-35 psu) (Yamaguchi et al. 1991) . Chatonella marina is highly resistant to harsh environmental conditions and even can survive under an irradiance of up to about 1000 µmol photons m −2 s −1 (Qiu et al. 2013) . Chatonella marina can produce a variety of metabolites such as neurotoxins, hemolysins toxins, and reactive oxygen, and thus the HABs triggered by this alga can usually cause mass mortality of fish (Shimasaki et al. 2021) . In recent years, great economic losses to aquaculture caused by C. marina-forming HABs have been reported in Korea (Kim et al. 2007 ), Japan (Onitsuka et al. 2011) , Europe (Waite and Lindahl, 2006) , and China (Wang et al. 2017b) . Therefore, it is important to establish efficient and sensitive detection methods for C. marina.

To date, the internationally recognized standards for the identification of microalgae are based on the morphological characteristics of the cells, including traditional microscopic observation and automatic image recognition by combining microscopic observation with computer technology. However, the traditional microscopic observation that is dependent on morphological structures has several limitations, such as low efficiency, poor timeliness, and high requirements for working personnel. Although these drawbacks have been overcome to some extent by automatic image recognition, this technique is not suitable for point-of-care testing (POCT) as it relies on flow cytometry and is therefore costly.

Since the 1980s, nucleic acid-based molecular techniques have been gradually applied to detect harmful algae. Several molecular detection techniques for HABs have been developed so far, including fluorescence in situ hybridization (FISH) (Miller and Scholin 2000; Liu et al. 2020) , sandwich hybridization (SH) (Diercks et al. 2008; Zhu et al. 2012) , polymerase chain reaction (PCR) (Lyu et al. 2017; Elleuch et al. 2020) , isothermal amplification (IA) (Toldrà et al. 2019a; Zhang et al. 2019) , microarrays (Taylor et al. 2013; Noyer et al. 2015) , biosensors (Diercks-Horn et al. 2011; Toldrà et al. 2019b) , and real-time qPCR (Nishimura et al. 2016; Zhang et al. 2016) . These molecular detection techniques have, to a certain extent, overcome the disadvantages of traditional morphological identification methods, greatly improved the detection efficiency, and provided new ideas for the monitoring and early warning of HABs.

The lateral flow strip is a relatively new molecular detection technique that has been applied in many different areas due to its simplicity and sensitivity. In the field of food detection, Suryoprabowo et al. (2021) achieved the semi-quantitative detection of tadalafil, an illicit drug in coffee samples, using a lateral flow immunoassay based on a fluorescently labeled monoclonal antibody. Yao et al. (2021) used chromatographic strips for the simultaneous detection of fipronil and its metabolites in eggs and cucumbers. In the medical field, Takarada et al. (2020) combined the chromatographic strip technique with loop-mediated isothermal amplification (LAMP) for the rapid detection of rifampicin-resistant Mycobacterium tuberculosis. Zhang et al. (2020) developed a lateral flow strip for the rapid detection of SARS-CoV-2 based on a double antibody sandwich principle, which can be used as a diagnostic aid for the COVID-19 pandemic. Based on the above examples, the lateral flow strip can be combined with a variety of nucleic acid detection techniques to achieve qualitative or even semi-quantitative detection results. Therefore, combining the lateral flow strip with existing detection techniques for HABs is a promising idea.

Up to now, the molecular detection methods targeting C. marina mainly include multiplex polymerase chain reaction (mPCR) (Sun et al. 2019) , quantitative polymerase chain reaction (qPCR) (Park et al. 2012) , hyperbranched rolling circle amplification (HRCA) (Nie et al. 2017) , loop-mediated isothermal amplification (LAMP) (Qin et al. 2019) , double probes rolling circle amplification (dpRCA) (Qin et al. 2020) , and membrane-based DNA array . To initially establish the lateral flow strip technique for the detection of C. marina, PCR amplification combined with the lateral flow strip was chosen in this study.

Although the lateral flow strip technique is efficient and intuitive for nucleic acid analysis, the preparation of lateral flow strips requires special instruments and is relatively costly, which can significantly increase the detection cost especially when the number of samples to be tested is large. Therefore, in this study, we developed a hand-prepared dot lateral flow strip (DLFS) and investigated its application to detect harmful microalgae in combination with PCR. To achieve these objectives, the PCR combined with DLFS (PCDS) was firstly developed by using the sequence of large subunit D1-D2 (LSU D1-D2) as the target gene. Secondly, the PCR system and the detection conditions of the DLFS were optimized. Finally, the specificity, sensitivity, and practicality of the developed PCDS were also evaluated.

Chatonella marina and control microalgal species used in this study are listed in Table 1 . These algal species were obtained by micropipette isolation, purchased from commercial companies, or kindly donated as gifts. The algal culture conditions were as follows: (1) the culture medium was f/2 (for diatoms) or f/2-Si medium (Guillard 1975 ) with a pH of 8.2 prepared from filtered and sterilized seawater;

(2) all of the algal cultures were grown at 20-24 °C, with a light intensity of 50-100 µmol photons m −2 s −1 , and a lightto-dark ratio of 12 h:12 h; and (3) all of the cultures were shaken regularly once or twice per day, with culture medium renewed every 2-3 weeks for good growth.

Aliquots (45 mL) of C. marina cultures in the logarithmic growth phase were subjected to centrifugation (4000 × g for 10 min at 4 °C), and then the resulting cell pellets were washed once under the same centrifugation conditions as above. Genomic DNA was extracted from the obtained algal cells using the Ezup Column Plant Genomic DNA Extraction Kit (Sangon Biotech, China). The concentration of the extracted genomic DNA solution (1.25 × 10 2 ng µL −1 ) was determined using a NAS-99 microspectrophotometer (Thermo Fisher Scientific, USA). The purity of genomic DNA was evaluated by the ratio of absorbance at 260 nm (A 260 ) to that at 280 nm (A 280 ). The obtained DNA samples were subjected to 1% agarose gel electrophoresis (AGE) and stored at − 20 °C.

The primers used for the PCDS assay consist of three parts, i.e., tag sequence, spacer, and specific sequence. The specific sequences for the forward and reverse primers were obtained from Sun et al. (2019) , and the tag sequences Tgn and Tsp, as well as the probes cTsp, Tgn, and cTgn, were obtained from Nagai et al. (2016) . All the probes and primers used in this study are summarized in Table 2 and were synthesized by Sangon Biotech (Shanghai) Co., Ltd. In particular, the gold nanoparticle probe cTgn was labeled with a sulfhydryl (SH) group at the 5′ end to allow it to bind to the gold nanoparticles. In addition, a poly (A 16 )-tail labeled with biotin was added to the 5′ end of the detection probe cTsp and the control probe Tgn. The purpose of the biotin labeling is to enable the probes to bind to the anti-biotin antibody to form large molecules for attachment to the detection membrane of the test strip, and the addition of the poly (A 16 ) tail is to prevent encapsulation of the probe sequence when the biotin binds to the anti-biotin antibody.

The schematic diagram principle of the PCDS is shown in Fig. 1 . The detection process consists of two stages, namely, PCR amplification and nucleic acid chromatography. In the PCR amplification phase, since both the forward primer Cm-Fp with Tgn and the reverse primer Cm-Rp with Tsp contain Spacer 9 that can effectively prevent the extension of DNA polymerase when the specific sequence triggers PCR amplification, they will produce the PCR products with double tags (Tgn and Tsp). In the nucleic acid chromatography phase, the PCR products are first mixed with chromatography buffer and added to the sample pad. Due to capillary action, the mixture flows laterally to the conjugate pad, where the tag sequence Tgn can be complementarily bound to the gold nanoparticlelabeled probe (cTgn), thus allowing the PCR products to form a complex with the gold nanoparticle. Next, the unbound gold nanoparticle-labeled probe and the complex mentioned above continue to laterally flow to the test For the practical application of the PCDS, 7.5 μL of PCR amplification products were first mixed with 92.5 μL of chromatography buffer. Then, the sample pad of the prepared DLFS was dipped into the mixture. After 5-10 min, the detection result was determined by observing whether red dots can be produced at the test and control dot sites or not. A positive result is determined when two red dots are produced at both the test and control dot sites, and a negative result is determined when only one red dot is produced at the control dot site. If no red spot appears in both the test and control spot sites, the test strip detection system is proven to be not working effectively.

The glass fiber SB06 (Shanghai Liangxin Technology Co., Ltd.) used as the sample pad was immersed in sample pad buffer (0.05 M Tris-HCl, 0.15 M NaCl, 0.25% Triton X-100; pH 8.0) for 20 min, dried at 37 °C for 2 h and then stored at room temperature (RT).

The solution containing gold nanoparticles with an average diameter of approximately 30 nm was prepared by the citrate reduction method (Turkevich and Enüstun 1963; Yeh et al. 2012) . Aliquots (1 mL) of gold nanoparticle solution were centrifuged at 12,000 × g for 20 min at RT. After removing the supernatant, 6 μL of 100 μM gold nanoparticle probe (cTgn) and 94 μL of 1 × TE buffer were added to the precipitate, mixed well, and incubated at 4 °C for 12 h. An equal volume (100 μL) of PBS solution (0.2 M NaCl, 20 mM Na 2 HPO 4 /NaH 2 PO 4 ; pH 7.0) was added, mixed quickly, and incubated at 4 °C for 24 h. The solution was centrifuged at RT for 10 min at 2000 × g. After removing the supernatant, the precipitate was resuspended with 200 μL of ultrapure water and centrifuged again under the same conditions to remove the unlabeled probes. Finally, the resultant gold nanoparticle-labeled probe was resuspended with 1 mL of embedding buffer (20 mM Na 3 PO 4 , 5% BSA, 0.25% Triton X-100, 8% sucrose) and stored at 4 °C away from light. When in use, the above-stored gold nanoparticle-labeled probe solution was first thoroughly dispersed by ultrasound under lightproof conditions, then uniformly sprayed onto glass fiber RB65 (Shanghai Liangxin Technology Co., Ltd.) (ca. 0.18 mL cm −2 ), dried at 37 °C for 2 h, and stored at RT.

The nitrocellulose membrane (NCM) (Sartorius UniSart CN140 Membrane) used as the detection membrane was successively immersed in ddH 2 O for 10 min and in 20 × SSC Technology) was mixed, respectively, with the test probe (cTsp) and the control probe (Tgn) (10 µM) with a volume ratio of 1 to 10, and aliquots (1 µL) of the mixture was spotted onto the test and control sites (approximately 7 mm apart). The spotted NCM was then UV cross-linked (UV cross-linker setting: wavelength, 254 nm; intensity, 90 J cm −2 ) for 2 min to bind the probe to the NCM firmly.

The schematic diagram of the assembly of DLFS is as shown in Fig. 2 . In brief, the detection membrane was first pasted on the PVC base (SM31-40) (Shanghai Liangxin 

Specific PCR was performed in a thermal cycler (Bioer Technology, China) with a total volume of 12.5 µL consisting of 1 × PCR buffer ( 

A total of 23 microalgal species (Table 1) , including the target species, were selected for a cross-reactivity test to verify the specificity of the established PCDS. In brief, universal PCR amplification was first performed using the genomic DNA from all the test species as templates with the previously reported primers D1 and D2 (Scholin et al. 1994) (Table 2) to determine whether the template quality was suitable for subsequent specific PCR with the primers Cm-Fp and Cm-Rp. Second, the same genomic DNA mentioned above was used as test samples for specific PCR and PCDS, respectively. The PCR products were analyzed by 2% AGE, and the results of the PCDS were judged by observing the presence or absence of colored dots on the test and control spot sites of DLFS.

The sensitivity of PDCS was tested using the genomic DNA of C. marina as a template. The genomic DNA prepared in the subsection "DNA extraction" was diluted tenfold with nuclease-free water (Sangon Biotech (Shanghai) Co., Ltd.) to prepare DNA test samples in a gradient of seven concentration dilutions (1.25 × 10 2 -1.25 × 10 -4 ng µL −1 ). The test samples were analyzed by PCDS, with the specific PCR as control. The specific PCR was performed with primers Cm-Fp and Cm-Rp. The PCR products were analyzed by 1.5% AGE, and the results of the PCDS were determined by observing the coloration results of DLFS.

The natural seawater that was used to prepare spiked samples was collected from Jinhai Bay (Weihai, Shandong, China) in the Yellow Sea, in which no microalgal target cell could be observed by microscopic examination. Aliquots of C. marina cultures in the exponential growth stage were fixed with 1.5% Luger's solution, and the cell concentration was determined by counting with a plankton counting chamber under light microscopy. The C. marina cells that were collected from 50 mL of algal culture by centrifugation (4 °C, 4000 × g, and 10 min) were resuspended in 50 mL of natural seawater, which was used as the initial spiked sample with a concentration of 10 5 cells mL −1 . Then, the initial sample was tenfold diluted with natural seawater to result in a series of spiked samples with the concentration range of 10 5 -10 −1 cells mL −1 . Algal cells were, respectively, Fig. 2 Schematic diagram of the structure of DLFS collected from these spiked samples by centrifugation using the same conditions as above, and DNA extracts were prepared using One-tube Plant Genome DNA Extraction Kit (Sangon Biotech, China) according to the manufacturer's instructions. The obtained crude DNA extracts were used as templates for specific PCR and the PCDS, respectively.

The PCR conditions, including annealing temperature, primer concentration, and Mg 2+ concentration were optimized to gain high amplification efficiency with a minimum yield of primer dimer. The optimization results of annealing temperature are shown in Fig. S1 . When the annealing temperature was higher than 63 °C, no target band could be detected by AGE, indicating that the PCR could not occur. In comparison, when the annealing temperature was between 55 and 59 °C, clear target bands could be detected by AGE, indicating that efficient PCR could occur. Therefore, the optimal annealing temperature was finally set at 55 °C to reduce primer dimers. Next, the primer concentration was optimized by setting the annealing temperature at 55 °C (Fig. S2) . Successful PCR amplification could occur with all primer concentrations. In addition, a much higher amplification efficiency could be detected with the primer concentration of 0.15, 0.20, 0.25, and 0.30 μM, respectively. The optimal final primer concentration was set to 0.15 μM to save costs and reduce primer dimer. Finally, the Mg 2+ concentration was optimized by setting the annealing temperature at 55 °C and the primer concentration at 0.15 μM (Fig. S3) . Because a much higher amplification efficiency could be detected with the Mg 2+ concentrations of 1.5, 2.0, and 2.5 mM, respectively, the optimal Mg 2+ concentration was set to 1.5 mM to save costs.

Based on the above results, the optimal PCR conditions (Table 3) were applied to perform PCR amplification (Fig. 3A) . The resultant PCR products were subjected to DLFS (Fig. 3B) in triplicate by setting the probe amount at both test and control spot sites to 10 pM. Three independent tests consistently showed that although DLFS could work well, i.e., the positive samples could produce red dots at both test and control spot sites of the strips, the blank control samples could also unexpectedly produce two light red dots at both test and control spot sites of the strips, which is actually a "false positive" result. Therefore, to further eliminate the "false positive" results, three parallel PCDS tests were performed by adjusting the amount of test probe to 7.5 pM (Fig. 3C) . Fortunately, all the tests produced the same results A AGE analysis of PCR products; B PCDS analysis using the preliminarily prepared DLFS by setting the probe amount to 10 pM; C PCDS analysis using the optimized DLFS by setting the probe amount to 7.5 pM. M, DL 1000 DNA marker; lanes 1, 3, and 5 are PCR products; lanes 2, 4, and 6 are blank controls that indicate that "false positive" has been successfully eliminated, i.e., PCR amplification products could produce red dots at both test and control spot sites of DLFS, while blank control could produce red dots only at control spot sites.

A total of 23 marine microalgal species, including C. marina, were selected for the cross-reactivity test to confirm the specificity of the established PCDS assay. First, the universal PCR amplification of the LSU rDNA D1-D2 region using the genomic DNA of all the algal species as the template consistently showed positive results, indicating that the quality of the DNA template for each species could meet the requirements of the amplification test (Fig. 4A) . Second, specific PCR amplification using the C. marina-specific primers Cm-Fp/Rp was carried out, showing that successful PCR amplification could exclusively occur with only the genomic DNA from the target species (Fig. 4B) . Finally, all the specific PCR amplification solutions (with or without PCR products) were subjected to a DLFS assay. The results showed that only the target species produced two red dots on the strips for test and control spot sites, whereas the non-target species and the blank control produced only one red spot on the strips for the control site (Fig. 4C) . In conclusion, the PCDS assay developed in this study is specific for the detection of the target algal species.

A series of tenfold dilutions of the target algal genomic DNA was used for comparative PCR-AGE and PCDS analysis using the specific primers Cm-Fp/Rp (Fig. 5) , respectively. PCR-AGE and PCDS displayed a detection limit of 1.25 × 10 -1 ng µL −1 and 1.25 × 10 −2 ng µL −1 for genomic DNA. Therefore, PCDS is 10 times more sensitive than PCR-AGE.

To assess the practicability of the established PCDS, spiked natural water samples containing target algal cells with the concentration range of 10 5 -10 −1 cells mL −1 were prepared and used as test samples for the comparative analysis by PCR-AGE and PCDS (Fig. 6) concentration ranges of 10 5 -10 2 cells mL −1 (Fig. 6A ) and 10 5 -10 1 cells mL −1 (Fig. 6B) , respectively. In other words, PCR-AGE and PCDS displayed a detection limit of 10 2 cells mL −1 and 10 1 cells mL −1 , respectively. Again, the tests with spiked samples indicated that PCDS is 10 times more sensitive than PCR-AGE.

Chatonella marina can produce several toxic metabolites such as reactive oxygen species (ROS), brevetoxin/brevetoxin-like compounds, free fatty acids (FFAs), hemagglutinins, and hemolysins (Shen et al. 2010) , which may play a synergistic role in ichthyotoxicity and indirectly cause large-scale fish mortality (Qiu et al. 2020 ). In addition, C. marina may exert an allelopathic effect on other dinoflagellates, triggering larger-scale HABs and causing serious economic and ecological damage to the aquatic environment (Fernandez-Herrera et al. 2016) . Therefore, it is important to establish a rapid detection method for C. marina to take effective measures to reduce costs before it causes serious damage.

In this study, we try to establish a convenient, sensitive, and practical detection method for C. marina. The existing molecular detection methods developed for C. marina including mPCR, qPCR, HRCA, LAMP, dpRCA, and membrane-based DNA array have been proven to be effective but still have some limitations. For example, mPCR and qPCR require relatively high requirements for primer design, and qPCR requires expensive fluorescent dyes, leading to a relatively high detection cost. Although the isothermal amplification techniques such as LAMP, HRCA, and dpRCA are independent of thermal cyclers, the need for the key components such as DNA polymerase with strand-displacement activity, Bst polymerase, and phosphorylated probes can also increase the cost of the assay. The membrane-based DNA array is not suitable for POCT because of the complex experimental process and long test time. By contrast, neither complex experimental procedures nor expensive components are required for PCDS. In addition, the cost for PCDS that is prepared by hand is much lower compared with the traditional chromatography strip technology, which makes PCDS more competent for POCT. The selection of suitable target genes is vital for a molecular detection method. Noncoding internal transcribed spacer (ITS), LSU rDNA, and small subunit (SSU) rDNA sequences are often selected as molecular markers for the discrimination of algal species. The main reason for this is that these genes contain both conserved and highly variable regions, facilitating the design of primers or detection probes specific to different algal species . Also, both ITS and LSU rDNA have been used as the main target for most of the established molecular detection methods for C. marina. For example, Park et al. (2012) designed the primers targeting ITS for specific qPCR detection of C. marina. Kai et al. (2006) established a single-cell PCR assay using the LSU rDNA as a target to distinguish between two Chattonella species. In this study, the LSU rDNA sequence of C. marina was selected as the target, and based on the previously designed specific primers (Sun et al. 2019) , the tag sequence was added and used for the establishment of the PCDS assay. The specificity experiments in this study in combination with previous studies again demonstrated the effectiveness of LSU rDNA for interspecies differentiation.

The overall optimization of the detection conditions is a crucial step in the development of an assay. According to the principle of PCDS, the production of primer dimers as a by-product of PCR can interfere with the determination of detection results, also known as "false positives." To eliminate the interference of "false positive," a series of control variables were optimized. First, the PCR conditions were optimized to avoid the production of primer dimer by successively determining the optimal annealing temperature (55 °C), primer concentration (0.15 μM), and Mg 2+ concentration (1.5 mM). However, DLFS analysis of blank control with the optimized PCR conditions showed that "false positives" could still interfere with specific detection. Therefore, we tried to adjust the amount (sampling volume) of the test probe on the strip and tested again. Fortunately, "false positive" for the blank controls was successfully eliminated in this way.

Sensitivity is a critical factor that could reflect the practical application value of a detection method. Therefore, in this study, we evaluated the detection limit of the established PCDS assay. First, a comparative test was performed using the serially diluted genomic DNA as test samples, showing that the sensitivity of PCDS is one order of magnitude higher than that of PCRAGE. Spiked natural water samples were further employed to assess the detection limit of PCDS, which was determined to be 10 cells mL −1 . In general, the sensitivity test with both the genomic DNA and the crude cell extracts consistently displayed that PCDS is an order of magnitude more sensitive than PCR-AGE. According to the previously reported results (Lee et al. 1995) , the early warning concentration for the outbreak of C. marina-forming HABs is 200 cells mL −1 , so the PCDS assay developed in this study can meet the practical needs. In addition, the sensitivity of PCDS is also comparable to that of the established molecular detection methods for C. marina including HRCA (Nie et al. 2017 ) and dpRCA-LFD (Qin et al. 2020) .

In conclusion, a novel method referred to as PCDS was developed for the highly specific and sensitive detection of C. marina in this study. The developed PCDS could enable the efficient detection of C. marina, with the intuitive results visible to the naked eyes. In addition, the low cost of preparation of DLFS also gives PCDS an advantage in terms of in situ detection, making it an effective means of preventing HABs. In the future, DLFS could be combined with RPA to further shorten the detection time and make it suitable for use in resource-poor areas to really meet the requirements for POCT. In addition, DLFS can also be combined with mPCR or multiplex recombinase polymerase amplification to realize the multiple detections of harmful algae.

The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s10811-021-02667-x.

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Data availability All data generated or analyzed during this study are included in this published article.